超灵敏同步检测多种真菌毒素的生物条形码免疫分析技术研究

Mycotoxins have posed a serious threat to the quality and safety of agricultural products. The current immunoassays still could not meet the requirement of monitoring of low-level and multicomponent mycotoxins simultaneously very well. To solve this technical bottleneck, the bio-bar code immunoassay with the capacity of ultrasensitivity and simultaneous detection will be developed to detect multicomponent mycotoxins in this research. Antibody-based magnetic nanoparticle probe will be prepared by coupling various kinds of monoclonal antibodies against mycotoxins (take AFB1, DON and ZEN, for example) with magnetic nanoparticle. Various antigen-based gold nanoparticle probes will be developed by the combination of different competitive antigens and corresponding bar code DNA with colloidal gold nanoparticles, respectively. Competitive immunoassays proceed between mycotoxins, antigen-based gold nanoparticle probes and antibody-based magnetic nanoparticle probe. After magnetic separation, the released bar code DNA will corresponding combine with detection probes and chip probes. The signal amplification achieve through the method of gold-label silver staining, detect with visual interpretation or scanning, and the simultaneous detection of multicomponent mycotoxins come true. The developed immunoassay based on bio-bar code assay will be optimized and evaluated, and applied to investigate the contamination of mycotoxins in agricultural products. Through carrying out this research, the method of ultrasensitive and simultaneous detection of multicomponent mycotoxins based on bio-bar code immunoassay will be developed, the new strategy of rapid, ultrasensitive and simultaneous detection of small molecular pollutants will be provided, the application of bio-bar code assay will be broadened, an important value will be showed in maintaining the quality and safety of agricultural products after its application.

真菌毒素严重威胁着农产品质量安全，现有免疫分析技术仍难以满足其低浓度混合污染同步检测需求。针对这一技术瓶颈，本项目拟开展基于生物条形码免疫分析的多种真菌毒素超灵敏同步检测技术研究。在磁性纳米颗粒表面同时修饰多种真菌毒素（以AFB1、DON和ZEN为例）单克隆抗体，制备磁纳米抗体探针；在胶体金纳米颗粒表面分别修饰不同竞争抗原和对应条形码DNA，制备多种金纳米抗原探针；上述两种探针与待测真菌毒素进行竞争性免疫反应，磁性分离；解离后的条形码DNA与检测探针和芯片探针对应结合；金标银染色法放大信号，可视化判读或扫描检测，实现多种真菌毒素同步检测；优化和评估新型检测技术，并应用于农产品真菌毒素污染调查研究。项目研究，将建立超灵敏同步检测多种真菌毒素的生物条形码免疫分析方法，提供小分子污染物快速超灵敏同步检测新策略，拓宽生物条形码技术应用范围，其推广应用对于保障农产品质量安全具有重要价值。

真菌毒素的简便快速、超高灵敏、同步多组分、高通量、现场检测技术开发对于保障农产品质量安全和人类健康具有重要现实意义。项目研究在真菌毒素抗原合成基础上制备获得的AFB1、DON、ZEN和OTA单克隆抗体，灵敏度IC50值分别为：0.254、25.2、0.59和0.91 ng/mL，并且具备高特异性。将新型标记、同步检测、生物条形码、可视化检测等多种策略有机结合，建立了基于酪胺信号放大技术的AFB1 酶免疫分析方法，其LOD和IC50值分别为0.004和0.039 ng/mL，灵敏度相比ELISA显著提升；研制了DON的ELISA试剂盒并应用于农产品污染调查研究，阳性检出水平在200.9-6480.6 ng/g；建立了OTA的半定量-定量免疫层析法分析检测技术，半定量LOD为0.5 ng/mL，定量LOD为0.1 ng/mL；开发了ZEN的生物条形码超灵敏免疫分析检测技术，LOD达到0.5 pg/mL，检测限低于ELISA法400倍；建立了生物条形码的AFB1和DON超灵敏免疫磁珠同步分析检测技术，实现pg/mL水平的超高灵敏度同步高效检测，相对传统ELISA方法LOD值分别降低480倍和245倍；开发了菌载体金颗粒生长增强型检测卡用于OTA和AFB1的同步筛查，完全抑制浓度分别降低20倍和12倍；此外建立的快速检测技术也应用于农副产品实际样本中真菌毒素污染调查评估。项目研究可拓宽生物条形码技术和新型功能化探针的应用范围，为农业环境食品质量安全提供检测技术储备，提供小分子污染物超灵敏同步快速检测新思路和技术体系。项目研究发表SCI论文7篇，其中第一标注4篇，第二标注3篇；授权并技术转让国家发明专利1项；培养毕业研究生3名，在读研究生3名；参与获得省级科学技术二等奖1项。